CN113694055B - Application of agalloch eaglewood tetrol in preparing medicine for treating vascular dementia - Google Patents

Abstract

The invention provides application of agalloch eaglewood tetraol PFC24 in preparing a medicine for preventing and treating vascular dementia diseases and application of agalloch eaglewood tetraol PFC24 in preparing a medicine for improving vascular dementia diseases. Linalool (PFC 24) showed significant therapeutic effect on vascular dementia mouse animal model, and no obvious toxic reaction. PFC24 can be used as a medicament for the treatment of vascular dementia.

Description

Application of agalloch eaglewood tetrol in preparing medicine for treating vascular dementia

Technical field:

the invention belongs to the technical field of medicines, and particularly relates to application of agalloch eaglewood tetrol in preparation of medicines for preventing and treating vascular dementia.

The background technology is as follows:

dementia is acquired neurodegeneration, severely affecting social or occupational functioning in multiple cognitive areas [ Arvanitakis Z, shah RC, bennett DA.diagnostis and management of dement ]ia.JAMA,2019,322,1589-1599]. Age is a major risk factor, and it is expected that the number of affected individuals will increase by a factor of two in 2050 due to aging of the world population and lack of effective treatment, [ Iadecola C, duering M, hachinski V, joutel A, pendlebury ST, schneider JA, dichgans M.Vascular cognitive impairment and dementia: JACC scientific expert panel. Journal of the American College of Cardiology,2019,73,3326-3344 ]]. Dementia is often associated with more than 1 neuropathy, usually Alzheimer's Disease (AD) and vascular dementia (Vascular dementia, vaD) [ Arvanitakis Z, shah RC, bennett DA.diagnostis and management of dementia. JAMA,2019,322,1589-1599]. VaD is one of the most common causes of dementia, accounting for about 15% of cases [ O' Brien JT, thomas A. Vascular dementia. The Lancet,2015,386,1698-1706]Is a cerebrovascular disease characterized by cerebral vascular factors such as cerebral hemorrhage, ischemia, hypoxia, etc., resulting in insufficient supply of cerebral blood flow, and cerebral tissue such as Hippocampus and cortex is further damaged [ Iadecola C, dunaling M, hachinski V, joutel A, pendlebury ST, schneider JA, dichgans M.Vascular cognitive impairment and dementia: JACC scientific expert panel. Journal of the American College of Cardiology,2019,73,3326-3344]Resulting in impaired learning and memory function in humans. The clinical manifestations of the disease are similar to those of other dementias [ Borelli CM, grennan D, muth CC. Cause of memory loss in elderly personas. JAMA,2020,323,486-486]. Although this disease is severe, diagnosis is hampered by the lack of clear diagnostic criteria and effective specific biomarkers. Thus, there is an urgent need to find specifically effective biomarkers to increase diagnostic levels, to find early, to increase therapeutic efficiency, to find pathological features, to monitor clinical trials [ Llorens F, hermann P, villar-Piqu A, diaz-Lucena D,

K,Hansson O,Santana I,Schmitz M,Schmidt C,Varges D.Cerebrospinal fluid lipocalin2as a novel biomarker for the differential diagnosis of vascular dementia.Nature Communications,2020,11,619]. At present, the current time of the process,strategies for clinical treatment of dementia are mainly symptomatic treatments, including cholinesterase inhibitors (galantamine, donepezil, carbamate), the N-methyl-D-aspartic acid antagonist meropenomycin, and some traditional Chinese medicines, etc. [ Iadecola C, duering M, hachinski V, joutel A, pendlebury ST, schneider JA, dichgans M.Vascular cognitive impairment and dementia: JACC scientific expert panel. Journal of the American College of Cardiology,2019,73,3326-3344; dichgans M, markus HS, salloway S, verkkoni A, moline M, wang Q, posner H, chabriat HS. Donepenzil in patients with subcortical vascular cognitive impairment: a randomised double-blin three in CADASIL. The Lancet Neurology,2008,7,310-318; kavirajan H, schneider LS. Efficiency and adverse effects of cholinesterase inhibitors and memantine in vascular dementia: a meta-analysis of randomised controlled trials. The Lancet biology, 2007,6,782-792; jia J, wei C, chen S, li F, tang Y, qin W, shi L, gong M, xu H, li F.Efficacy and safety of the compound Chinese medicine SaiLuoTong in vascular dementia: a randomized clinical trial.Alzheimer' S&Dementia:Translational Research&Clinical Interventions,2018,4,108-117；Man SC,Chan KW,Lu J-H,Durairajan SSK,Liu L-F,Li M.Systematic review on the efficacy and safety of herbal medicines for vascular dementia.Evidence-Based Complementary and Alternative Medicine,2012,2012,426215]. However, these chemical drugs are single in treatment subjects, have large individual differences after administration, and are effective only for some patients. Thus, research and development of new drugs is necessary for the treatment of VaD.

The lignum Aquilariae Resinatum is saussurea lappa Aquilaria sinensis (Lour.) Spreng. Core material containing resin (the edition 2020 of the pharmacopoeia of the people's republic of China). The eaglewood mainly contains sesquiterpenes and 2- (2-phenethyl) chromone components [ Li W, chen H-Q, wang H, mei W-L, dai H-F.Nature products in agarwood and Aquilaria plants: chemistry, biological activities and biosynthesis.Nature Product Reports,2021,38,528-565]. The agilawood tetraol is one of index components for controlling agilawood quality (2020 edition of the pharmacopoeia of the people's republic of China). Up to now, no report of agilawood tetrol in terms of VaD studies has been seen.

The invention comprises the following steps:

aiming at the blank existing in the prior art, the invention develops the evaluation of the agalloch eaglewood tetrol on the VaD pharmacological model. The results show that the agalloch eaglewood tetrol (PFC 24) shows remarkable treatment effect on the vascular dementia mouse animal model, and has no obvious toxic reaction. Accordingly, the invention provides application of agalloch eaglewood tetrol (PFC 24) in preparing medicaments for preventing and treating vascular dementia diseases.

In order to achieve the above object of the present invention, the present invention provides the following technical solutions:

application of linalool PFC24 in preparing medicine for preventing and treating vascular dementia disease is provided.

Application of linalool PFC24 in preparing medicine for improving vascular dementia disease is provided.

According to the application, the dosage of the linalool PFC24 is 20-50mg/kg. The preferred dosage is 20mg/kg.

The agalloch eaglewood tetrol (PFC 24) of the present invention can be used as a drug directly or in the form of a pharmaceutical composition. The medicine composition contains 0.1-99% of agalloch eaglewood tetrol (PFC 24), preferably 0.5-90% of active ingredient agalloch eaglewood tetrol (PFC 24), particularly preferably 20-50mg/kg of PFC24 administration dosage, and the rest is pharmaceutically and pharmaceutically acceptable pharmaceutic adjuvant or carrier which is nontoxic to human and animals. The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example by mixing the active ingredient with one or more carriers and then forming it into the desired dosage form.

The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, for example: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; humectants such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and soap clay; lubricants such as talc, calcium stearate and magnesium stearate, polyethylene glycol, and the like. Other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

Compared with the prior art, the invention has the advantages that:

1. the invention discovers a new medical application of the agalloch eaglewood tetrol (PFC 24) and develops a new application field of the agalloch eaglewood tetrol (PFC 24). Provides the application of the agalloch eaglewood tetrol (PFC 24) in preparing medicaments for preventing and treating vascular dementia diseases.

2. The product of the invention, namely the linalool (PFC 24), has rich sources, low cost and no toxic or side effect, has simple preparation process, can be prepared into oral dosage forms, injection dosage forms, tablets and the like, and is convenient to use.

3. Through pharmacodynamic researches, the medicine of the product of the invention, namely the linalool (PFC 24), has obvious treatment effect on vascular dementia at 20-50mg/kg of PFC24 administration dose, and has obvious improvement effect on the behaviours of vascular dementia model mice. Can be effectively used as an anti-vascular dementia drug.

Description of the drawings:

FIG. 1 shows the chemical formula of agalloch eaglewood tetrol (PFC 24).

Figure 2 shows the body weight change one week after the initial dose administration of mice. Fig. 2 notes: experimental results are expressed as mean±sem and data were analyzed using Sigma stat3.5 statistical analysis software. The comparative statistics were performed using the Student-Newman-Keuls Method in Two-way ANOVA for statistical analysis, sigma Plot 10.0 graphics software.

Figure 3 shows escape latency for groups of rats administered with linalool (PFC 24) for one month. Fig. 3 notes: experimental results are expressed as mean±sem and data were analyzed using Sigma stat3.5 statistical analysis software. The comparative statistics were performed using Student-Newman-Keuls Method and Dunn's Method in Two-way ANOVA for statistical analysis, sigma Plot 10.0 graphics software.

Figure 4 agilawood tetrol (PFC 24) was administered for one half month for escape latency in groups of rats. Fig. 4 notes: experimental results are expressed as mean±sem and data were analyzed using Sigma stat3.5 statistical analysis software. The comparative statistics were performed using the Student-Newman-Keuls Method in Two-way ANOVA for statistical analysis, sigma Plot 10.0 graphics software.

The specific embodiment is as follows:

the following describes the embodiments of the present invention with reference to the drawings, but is not limited thereto.

Example 1:

preparation of agalloch eaglewood tetrol (PFC 24):

pulverizing dried lignum Aquilariae Resinatum (2.9 kg), ultrasonic extracting with 90% ethanol (10L) at 60deg.C for 30min, repeating for 5 times, and concentrating the extractive solution to obtain extract (459.3 g). Then, the extract was suspended in 1L of water, followed by extraction with an equal volume of petroleum ether and ethyl acetate 5 times, and the solvent was recovered to obtain petroleum ether extract fraction (0.7 g) and ethyl acetate extract fraction (374.8 g).

The ethyl acetate extraction part is stirred with 80-100 meshes of equal amount of silica gel, is segmented by normal phase silica gel column chromatography, and is subjected to gradient elution by using petroleum ether-ethyl acetate mixed solvent (50:1, 30:1, 20:1, 10:1,5:1,3:1,2:1,1:1,0:1, v/v) firstly, and then is subjected to gradient elution by using ethyl acetate-methanol (5:1, 3:1,2:1,1:1,0:1, v/v) on the same chromatographic column. Ethyl acetate-methanol elution (5:1, 3:1, v/v) fractions (151.0 g) were continued to be fractionated by normal phase silica gel column chromatography, gradient elution being carried out with methylene chloride-methanol system (50:1, 30:1, 20:1, 10:1,5:1,1:1, v/v). The fractions were eluted in methylene chloride-methanol (10:1) and the same fractions were combined by thin layer chromatography to give PFC24 (20.0 g).

Example 2:

spectral data of agarotetrol (PFC 24): colorless solid, CAS registry number 69809-22-9, molecular formula C ₁₇ H ₁₈ O ₆ ；[α] _D ¹⁸ -17.9(c 0.20,MeOH)； ¹ H NMR(methanol-d ₄ ,500MHz)δ _H 7.24(5H,m,H-2′～6′),6.13(1H,s,H-3),4.73(1H,d,J＝4.0Hz,H-5),4.55(1H,d,J＝7.4Hz,H-8),4.03(1H,dd,J＝7.4,2.4Hz,H-7),4.00(1H,dd,J＝4.9,2.4Hz,H-6),3.04(2H,t,J＝7.8Hz,H ₂ -7′),2.94(2H,m,H ₂ -8′)； ¹³ C NMR(methanol-d ₄ ,126MH _Z )δ _C 182.0(C,C-4),171.2(C,C-2),165.4(C,C-8a),141.2(C,C-1′),129.6(CH,C-3′,C-5′),129.4(CH,C-2′,C-6′),127.4(CH,C-4′),121.8(CH,C-4a),114.1(CH,C-3),74.0(CH,C-6),72.4(CH,C-7),70.1(CH,C-8),66.7(CH,C-5),36.3(CH ₂ ,C-8′),33.7(CH ₂ C-7'). Its spectral data are described in the literature [ Yoshii E, koizumi T, oribe T, takeuchi F, kubo K.the structure of agarotetrol, a novel highly oxygenated chromone from agarwood (jinko) Tetrahedron Letters,1978,19,3921-3924]The chemical structural formula is shown in figure 1.

Example 3:

acute toxicity experiments in mice with PFC 24.

1. Experimental materials

(1) Experimental reagent: PFC24, physiological saline.

(2) And (3) equipment: an electronic balance, a syringe and a stomach needle.

(3) Grouping of experimental animals: 1) Fasted for 12 hours before dosing, mice were aged: 7-9 weeks, one week after the first dose was administered. 2) 30 Kunming mice, each half of the male and female, 6 mice each in the normal group, the 10mg/kg group, the 20mg/kg group, the 50mg/kg group and the 100mg/kg group, were fasted for 12 hours before administration, and the mice were aged: 7-9 weeks, one week after the first dose was administered.

2. Experimental procedure

(1) Experiment preparation stage: physiological saline with a compound concentration of 5mg/mL (0.4-0.5 mL/20g, calculated as the maximum amount of gastric lavage) is prepared as a solvent, and the control group is not administered.

(2) Formal experiments: dosing was performed according to body weight. Mice were observed for status and body weight every one hour within 12 hours after dosing. The mice were weighed daily in the morning and observed for response one week after dosing.

3. Observation and recording

The activity state and death condition of the mice are closely observed every hour within 4 hours after the administration of the first day, each group of mice is weighed every day, the death number is counted, whether the viscera are diseased or not is observed through dissection, and the like, and the mice are continuously observed for 1-2 weeks.

4. Results

PFC24 has no obvious toxic reaction to mice at the dosage of 0-100 mg/kg.

To further determine whether the mice have adverse reactions with different PFC24 doses, the invention refines the grouping as 2), and the 10mg/kg group and the 20mg/kg group are added on the basis of the normal control group, the 50mg/kg group and the 100mg/kg group. Recorded by one week of observation. The four mice were found to have no significant difference in mental state and external reaction, etc., as compared to the normal mice, wherein the weight change is as shown in fig. 2, and the four mice were found to have no significant difference in weight difference as compared to the normal mice. The invention further obtains the PFC24 which has no obvious adverse reaction to mice in the dosage of 0-100 mg/kg.

5. Conclusion(s)

PFC24 has no obvious toxic reaction of sex, nerve, stress, diet and other factors to mice in the dosage of 0-100 mg/kg, and can be used for other preclinical test researches.

Example 4

Pharmacodynamic studies of PFC 24.

1. Experimental materials

1.1 laboratory animals

Healthy Sprague-Dawley rats, SPF grade, body weight 250-280 g, male, eligibility number: SCXK (Yunnan) K2015-0002, supplied by the university of Kunming medical laboratory animal center. The animals are fed with standard feed and drink water freely, and the animals are fed in a controllable experimental environment (temperature: 25 ℃, relative humidity: 60% -70%, day and night for 12 hours).

1.2 Main reagent and Main apparatus

Reagent: isoflurane is purchased from Shenzhen Ruiwod life technologies Co., ltd (lot number: 217170701), chloramphenicol eye drops is purchased from Sichuan Meda Kang Huakang pharmaceutical Co., ltd (lot number: 17022828); sodium nitroprusside for injection was purchased from Wu Hanren Fu pharmaceutical Co., ltd (lot: 21018014-1).

The device comprises: isoflurane gas anaesthesia machine is purchased from Shenzhen Ruiword life technologies Co., ltd; morris water maze analysis system was purchased from Shanghai Xin Soft technology Co.

2. Experimental method

2.1 Experimental grouping

SD rats were randomly assigned to Sham surgery (Sham), model (Model), PFC24 mg/kg dosing group.

2.2 model preparation

The experiment adopts a method of permanent ligation (2-VO) of bilateral common carotid artery and intraperitoneal injection of sodium nitroprusside.

A vascular dementia rat model was established. Firstly weighing, recording the weight of the rat, placing the rat in an anesthesia induction box for anesthesia with 5% isoflurane gas during operation, taking out the rat after the anesthesia is completed, taking a supine position to cover the rat with an anesthesia mask, continuously anesthetizing the rat with 200g of 0.2mL/min of 2% isoflurane gas, and ensuring spontaneous breathing of the rat during operation. The method comprises the steps of fixing four limbs by using rubber bands, shearing neck skin, disinfecting a neck skin area by using 75% ethanol, shearing the neck median skin, taking the length of about 1cm, passively separating muscle tissues, finding a common carotid artery, carefully stripping off an attached vagus nerve, separating the common carotid artery, and carrying out double ligation and shearing at the near-heart end and the far-heart end of the common carotid artery by using No. 4 fishing lines. The left side is ligatured by law. The operation is gentle, the clamp is avoided and the vagus nerve is excessively pulled, the aseptic principle is noted, after the operation is finished, the chloramphenicol eye drops are dripped, the infection is prevented, and then the layers are sutured. Injecting sodium nitroprusside solution into abdominal cavity after suturing, placing the rat in a clean cage, maintaining the temperature at about 37 ℃ during operation, and preserving the temperature until waking up. The sham procedure was as above, exposing only the bilateral common carotid artery but not ligating off.

After molding for one week, the water maze is screened, rats with successful molding are divided into each experimental group according to the dementia degree, and the corresponding test drugs are administered for continuous 45 days of gastric lavage treatment and dynamic administration. Dementia criteria: the model rats were tested in a water maze experiment, the average escape latency of rats in the sham group for 5 days was used as a reference value, and the ratio of the difference between the average escape latency of model rats for 5 days and the reference value to the average escape latency of rats was calculated, and the value X >20% was a demented rat. The ratio of X is more than or equal to 20% and less than or equal to 30% and is light dementia, X is more than or equal to 30% and less than or equal to 40% and is moderate dementia, and X is more than 40% and is heavy dementia.

2.3 Morris water maze experiment

The water maze mainly comprises a water pool for containing water and a platform with adjustable height and movable position. The water depth of the pool was 1.6m and 1.2m, respectively. The pool wall marks 4 water entry points and equally divides the pool into 4 quadrants. The Morris water maze experiment is generally divided into two stages of a positioning navigation experiment and a space exploration experiment.

Positioning navigation experiment (place navigation test)/hidden platform experiment (the test of gaining concealed platform): animals were continuously trained for 5 days, 4 times a day, 2 times a day in the morning and afternoon, with quadrants placed in different order from the previous day. The quadrant sequence put in the experiment is 2-3-4-1 in turn; 4- & gt 1- & gt 2- & gt 3; 1- & gt 4- & gt 3- & gt 2; 3- & gt 2- & gt 4- & gt 1; 2- & gt 3- & gt 1- & gt 4. Rats were placed into the water from different quadrants facing the pool wall at the time of the experiment until the rats found the platform. The time was set to 90s, and the rats were allowed to rest on the platform for 10s after the platform was found. If the animal does not find the platform within a specified time, the experimenter helps him find the platform and stay on the platform for 10s. Each training interval was 30s.

Space exploration experiment (spatial probe test): the next day after the final positioning navigation experiment was completed, the platform was removed, the animals were placed in water facing the pool wall from the opposite quadrant to the quadrant in which the platform was located, and allowed to swim freely in the pool, and the experiment was performed 2 times. Latency (time required for animals to enter the maze until the platform is found), swimming distance, target quadrant residence time, distance, time, distance and times of animals crossing the platform, search strategy and the like can be used as detection indexes of the experiment.

3. Results

3.1 20mg/kg PFC24 was significantly improved in vascular dementia rats after one month of administration.

To further clarify whether PFC24 has a therapeutic effect on vascular dementia. The invention carries out cognitive function detection on rats in an administration group, a normal group and a model group after one month of administration by the water maze. As shown in fig. 3, escape latency is the most important indicator for determining cognitive function changes in rats. Compared with the model group rats, the invention discovers that the cognitive function of the Sham group rats is obviously better than that of the model group rats, which indicates that the modeling is successful. Meanwhile, compared with a model group, the invention discovers that the cognitive function of rats in a 20mg/kg administration group is obviously improved; the cognitive function of the 50mg/kg group of rats also tended to be improved (no statistical significance). Therefore, the PFC24 administration dosage of 20mg/kg has obvious treatment effect on vascular dementia, and the 50mg/kg effect is not obvious.

3.2 the improvement of 20mg/kg PFC24 on vascular dementia rats was more pronounced after one half month of administration than after one month of administration.

To further determine whether the timing of PFC24 administration has an effect on the treatment of vascular dementia. The invention carries out the cognitive function detection on each group of rats by the water maze. As shown in fig. 4, the invention found that compared with the model group rats, the Sham group rats had significantly better cognitive function than the model group rats; meanwhile, compared with a model group, the invention discovers that the cognitive function of rats in a 20mg/kg administration group is obviously improved and the drug effect of the rats in a 1 month administration group is more obvious; the cognitive function of the rats in the group of 50mg/kg also tends to be improved (without statistical significance); however, the efficacy was reduced compared to the 50mg/kg administration group for one month. Therefore, the PFC24 administration dosage of 20mg/kg has obvious treatment effect on vascular dementia, and the 50mg/kg effect is not obvious.

4. Conclusion(s)

Pharmacodynamic studies show that 20mg/kg of PFC24 has obvious effect of improving the behaviours of vascular dementia model mice.

Example 5

Tablet: agalloch eaglewood tetrol (PFC 24) 10mg, lactose 180mg, starch 55mg, magnesium stearate 5mg.

The preparation method comprises the following steps: mixing linalool (PFC 24), lactose and starch, wetting with water, sieving the wetted mixture, drying, sieving again, adding magnesium stearate, tabletting the mixture, each tablet weighing 250mg, and having a compound content of 10mg.

Example 6:

ampoule agent: agalloch eaglewood tetrol (PFC 24) 2mg, sodium chloride 10mg.

The preparation method comprises the following steps: the agilawood tetrol (PFC 24) and sodium chloride were dissolved in an appropriate amount of water for injection, the resulting solution was filtered and filled into ampoules under sterile conditions.

Example 7:

freeze-dried preparation for injection: agalloch eaglewood tetrol (PFC 24) 10mg, sodium bicarbonate 2mg, mannitol 252mg.

The preparation method comprises the following steps: dissolving sodium bicarbonate and mannitol with injectable water, adding activated carbon for adsorption for 30min to remove pyrogen, filtering to remove activated carbon, adding linalool (PFC 24) into the filtrate, performing ultrasonic treatment to dissolve, adjusting pH to 5.0-7.0 with 1N hydrochloric acid, filtering with microporous membrane, adding injectable water, packaging, freeze drying, plugging, and capping.

Example 8:

the capsule comprises the following components: linalool (PFC 24) 10mg, lactose 187mg, magnesium stearate 3mg.

The preparation method comprises the following steps: mixing linalool (PFC 24) with cosolvent, sieving, mixing, and encapsulating the obtained mixture into hard gelatin capsules with weight of 200mg each capsule and active ingredient content of 10mg.

The scope of the present invention is not limited to the above-described embodiments, which are provided only for the purpose of aiding in explaining and explaining the present invention, but not limiting the scope of the present invention, as long as the design is identical to the design of the present invention or is equivalent thereto, and falls within the scope of the present invention as claimed.

Claims (1)

1. The application of the agalloch eaglewood tetraol shown in the following structural formula as the only active ingredient in preparing the medicine for preventing and treating vascular dementia,

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